Genomics and The Future of Medicine Gene chip diagnoses and designing ourselves

T Mueller

Division of Nephrology and ImmunologyUniversity of Alberta

Technology and Future of Medicine CourseNov 02, 2011

Leonard Cohen and Medicine

Thomas F MuellerDivision of Nephrology and Immunology

University of Alberta

International Leonard Cohen FestivalWestin HotelJuly 24, 2008

Leonard Cohen Medicine

leonardcohenforum.com • Search25 Jan 2008 ... Leonard Cohen, A Life In Art. I often wondered whether Leonard had ... and Dr. Thomas Mueller who will speak on "Leonard Cohen's Medicine". ... www.leonardcohenforum.com/search.php?keywords=nadel - 32k -

Leonard Cohen Medicine

Topics1. Background

• Human Genome Project• Gene Expression

2. Techniques• PCR• Microarray• Sources• Analysis

3. Application • Strengths and weaknesses• Outcome

4. Summary and Outlook• OGOD• Integration of data• Clinical phenotyping

Initial sequencing and analysis of thehuman genome

International Human Genome Sequencing ConsortiumNature 2001, VOL 409, 15 FEBRUARY 2001

Science 16 February 2001: Vol. 291 no. 5507 pp. 1304-1351

The Sequence of the Human GenomeJ Craig Venter et al.

The Human Genome Project

• book of life• would strangle research• genome race between public and private• White House announcement June 2000• Wall Street promises of genetic crystal balls

The Human Genome Project

• book of life• would strangle research• genome race between public and private• White House announcement June 2000• Wall Street promises of genetic crystal balls

• cancer (most diseases) not yet cured• overall disappointment• …

How many genes are in the human genome?

Genome experts … not even close …Science 2007; 316: 1113

How many genes are in the human genome?

… about 20,000 protein-coding genes …

Science 2007; 316: 1113

The Human Genome Project

• greatest impact of genomics• to investigate biological phenomena in a

comprehensive, unbiased, hypothesis-free manner

• in basic biology understanding of • protein-coding genes• non-coding genes• regulatory sequences

• in medicine to discover• gene and pathways underlying diseases• about 2,850 genes underlying Mendelian diseases• about 1,100 loci affecting common polygenic disorders• and about 150 new recurrent targets of somatic mutation in cancer

• costs of DNA sequencing plummeted by around 100,000 fold• RNA-Seq is also being applied to RNA transcripts to count their abundance• sequencing will become a routine tool to characterize

• patient’s individual genomes• cancer genomes• immune repertoires, and• microbiomes

Where is molecular profiling today?

Where is molecular profiling today?

ease of implementation

bene

fit to

the

heal

th

piece of cakegoing to the moon would be easier

zilc

hra

dica

l

My personal experience and journey …

Where is molecular profiling today?

ease of implementation

bene

fit to

the

heal

th

piece of cakegoing to the moon would be easier

zilc

hra

dica

l

2004

2011

Naesens, Sarwal, Nat Rev Practice Nephro 2011

Naesens, Sarwal, Nat Rev Practice Nephro 2011

Topics1. Background

• Human Genome Project• Gene Expression

2. Techniques• PCR• Microarray• Sources• Analysis

3. Application • Strengths and weaknesses• Outcome

4. Summary and Outlook• OGOD• Integration of data• Clinical phenotyping

DNA synthesis(replication)

RNA synthesis(transcription)

protein synthesis(translation)

DNA

RNA

Protein

amino acids

reverse transcription

polymerase chain reaction

continuously monitored

real-time RT-PCR

PCR – an alternative to measure transcription

1 1

2 2

3 4

4 8

5 16

6 32

7 64

8 128

9 256

10 512

11 1024

12 2048

13 4096

14 8192

15 16384

16 32768

…

49 2.81475E+14

50 5.6295E+14

51 1.1259E+15

52 2.2518E+15

53 4.5036E+15

54 9.0072E+15

55 1.80144E+16

56 3.60288E+16

57 7.20576E+16

58 1.44115E+17

59 2.8823E+17

60 5.76461E+17

61 1.15292E+18

62 2.30584E+18

63 4.61169E+18

64 9.22337E+18

amplification

DNA synthesis(replication)

RNA synthesis(transcription)

protein synthesis(translation)

DNA

RNA

Protein

amino acids

cDNA synthesis

transcription and labelling

oligonucleotide microarrays

hybridization and staining

Principle of microarrays

Affymetrix® info material

Principle of microarrays

Affymetrix® info material

Principle of microarrays

Affymetrix® info material

Looking at the stars

Baby steps

Bittner, Meltzer, Trent. Nature Genetics 1999; 22: 213.

In the film What About Bob … Bill Murray plays a mental patient with a multi-phobic personality whose fear of almost everything leaves him in a constant state of panic. …

… an eminent psychiatrist whose therapy goals for Bob are summed up in his book entitled Baby Steps, in which he advocates setting small, reasonable goals one day a time – one tiny step at a time.

The multi-phobic character will resonate all too well with the geneticist who faces the rapid evolution of bioinformatics driven by massive quantities of data produced by genome-scale technologies. This technology brings us face to face with the underlying complexity of biological systems and genome-wide function … and the key to success is cross-disciplinary collaboration –and effective communication – with those who develop data analysis and integration tools - the mathematicians.

Data analysis and integration: of steps and arrows

Matrix of transcript levels

Signature

Profile

Gene 1Gene 2

.

.

.

Gene M

Sample 1 Sample 2 . . . Sample N

Visualization and analysis of transcript profiles by heatmaps, dendrograms, and clusters

Bittner, Meltzer, Trent. Nature Genetics 1999; 22: 213.

Bittner, Meltzer, Trent. Nature Genetics 1999; 22: 213.

Analysis of microarray transcript data

Topics1. Background

• Human Genome Project• Gene Expression

2. Techniques• PCR• Microarray• Sources• Analysis

3. Application • Strengths and weaknesses• Outcome

4. Summary and Outlook• OGOD• Integration of data• Clinical phenotyping

Living donorkidneys (n=76)

Deceased donor kidneys (n=67)

1hour

Kidney biopsy

Microarrayanalysis

recipient

Clinical data

Clinical data

analysis

1-2 days

GC project

Renal scan(DTPA or MAG3)

HE stained implant biopsies of living and deceased donors

C

A

ED

B

Which kidney is from a deceased donor?

C

A

ED

B

deceased deceasedliving donors

x

x

The transcriptome reflects donor origin

Mueller T et al, Am J Transplant 2008

Cluster 2:

DD Low riskCluster 3:

DD High riskCluster 1:

LD

The kidneys are clustered in relation to risk of delayed graft function (DGF)

x

x

Mate kidneys cluster together

Transcript levels of 10 acute phase response genes in implant biopsies

CEW OAL

Distribution of all individual kidneys based on their transcriptome changes

“good”“bad”

‘Outliers’ indicated by the transciptome changes

“good”“bad”

BP ↓↓

DGF +++

sepsis

kidney heart

The same transcripts are increased in Heart, Liver, and Kidney during Rejection

liver

d5/Co Gene

1075 Gzmb

827 Serpina3g

273 Ccl3

174 Ms4a4b

126 Slfn1

118 AI447904

111 Bcl2a1a

93 Lpxn

90 Cd3d

88 Socs1

d5/Co Gene

162 Gzmb

122 Cd3d

108 Serpina3g

92 Cd3g

75 Cd8b

73 AI447904

66 Ctsw

58 Ms4a4b

56 Ms4a4b

55 Lat

d10/Co Gene

50 Pglyrp1

29 Gng2

29 Emb

24 Cd3d

20 Myo1g

20 Pdcd1

17 Ly9

13 Cd8b

12 Cd37

12 Ltb

287

150common

Mean ↑ 19-fold Mean ↑ 6-fold Mean ↑ 38-fold

Mueller, Einecke et al, Am J Transplant 2008.

The continuity of changes in kidney biopsies

Where is molecular profiling today?

Do we have achieved personalized medicine?

“… we simply cannot accurately predict outcomes for the most clinically relevant sample size – a single patient.”

B Kaplan and J Schold

Odysseus or Hamlet – that is now the question

Science 2005; 309: 2010.

Differences in phenotypes in fingerprints of identical twins and the first cloned cat

Michiels S et alPrediction of cancer outcome with microarraysLancet 2005; 365: 488-92

“Findings: The list of genes identified as predictors of prognosis was highly unstable; molecular signatures strongly depended on the selection of patients in the training sets. … Because of inadequate validation … overoptimistic results … five of the seven studies did not classify patients better than chance.”

“Microarrays and molecular research: noise discovery?”

Systems biology approach

Naesens, Sarwal, Nat Rev Practice Nephro 2011

What was the discovery that made Galileo Galilei a tenured Professor

A discovery of 4 satellites resolving about Jupiter B contradicting that the earth was the centre of all celestial motionsC detecting approaching war ships 2 hours earlier D placing the Sun at the centre of the universeE describing the uniformity of acceleration independent of mass